Among adolescents and college students, 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") use has steadily grown, while the use of many other stimulants has declined or stabilized. Recently, consumption has been rising among young women [74]. Consequently, there is a substantial risk of exposure among women who are, or become, pregnant while abusing MDMA. Recently, we have demonstrated that prenatal MDMA exposure from embryonic days 14-20 (E14-20) induced long lasting decreases in DA and 5-HT turnover and increases in tyrosine hydroxylase (TH) fiber density [42]. TH fiber density in prenatally MDMA exposed rats was significantly and dramatically increased in the prefrontal cortex (PFC: 502%) as well as in striatum (STR: 9%), nucleus accumbens (NAc: 8%) and the lateral septum (LS: 15%) at P21. Stereologic cell counts of TH+ neurons in the substantia nigra (SN) and ventral tegmental area (VTA) revealed no changes in cell number, suggesting that the increased fiber density is a result of axonal sprouting rather than increased cell numbers. We therefore hypothesized that target derived pro-growth cues, specifically axonal guidance molecules or trophic factors, are changed in subjects prenatally exposed to MDMA and that such cues are the mechanisms whereby MDMA achieves this significant morphological increase in TH fiber density. In support of this hypothesis we have demonstrated that prenatal MDMA results in significant mRNA and protein increases in the axonal guidance cues netrin-1 and ephrin-b2 within the striatum and nucleus accumbens. These molecules govern axon innervation patterns via regulation and orientation of axonal growth. Collectively, these findings are the first to demonstrate that prenatal MDMA exposure results in persistent alterations in the development of the neonatal brain. The aims of this application are designed to determine the extent and time course of the development of axonal guidance cues, trophic factors and consequent increases in TH fiber density in DA-rich target regions (specifically the striatum and prefrontal cortex) after prenatal MDMA exposure. In addition, by utilization of the brain slice culture system, we can examine the consequences of the direct application of MDMA on the expression of these guidance and growth cues in an in vitro source-target system. [unreadable] [unreadable] These studies will provide a sound basis for future experiments into the consequences of prenatal MDMA on the developing and aging brain. In addition, they will provide a basis for pharmacologic interventions aimed at minimizing the profound alterations in axonal sprouting, guidance and connectivity that we have already begun to observe experimentally. [unreadable] [unreadable]